Control of cell proliferation and differentiation by the Retinoblastoma tumor suppressor pathway

项目总结/摘要 视网膜母细胞瘤（Rb）肿瘤抑制通路的失活是视网膜母细胞瘤（Rb）的早期必然事件。 人类癌症Rb通路的关键靶点之一是E2 F转录家族 因子，其调节细胞增殖和凋亡。的重叠和冗余功能 E2 F和Rb肿瘤抑制蛋白（pRB）家族的哺乳动物成员是一个主要的肿瘤抑制蛋白。 在鉴定其体内功能方面存在障碍。果蝇等模式生物提供了 有吸引力的替代品，以补充哺乳动物系统中的研究，由于高度保守的 Rb通路和果蝇对遗传分析的顺从性。我们的研究计划旨在 在解决两个基本问题：什么是Rb途径的基本功能， Rb通路突变如何影响组织中不同的细胞类型？到 为了解决这些问题，我们正在使用遗传，基因组和单细胞RNA-seq方法。 我们已经确定了E2 F和Rb蛋白调节生长的成人骨骼肌， 肌原纤维形成，这对生存力很重要。我们将确定Rb通路是如何 调节肌肉发育过程中的基因表达和细胞代谢， 研究E2 F缺乏在特定组织（如成人）中推断的全身效应。 骨骼肌和脂肪体。我们已经调整了Drop-seq，一种单细胞RNA测序， 技术，以确定Rb途径突变在异质组织中的作用。我们有 通过建立第一个果蝇野生细胞图谱， 型眼影像盘。通过Drop-Seq分析Rb突变体揭示了细胞内 酸化在小细胞群体，使他们独特的敏感性凋亡。链路 将进一步评估细胞凋亡和酸化之间的关系。在其他问题中， 用Drop-seq解决的是为什么Hippo通路的失活会诱导 Rb突变型光感受器的去分化。总体而言，拟议的研究将提高 我们对pRB及其在动物发育过程中的功能的了解。

PROJECT SUMMARY/ABSTRACT Inactivation of the retinoblastoma (Rb) tumor suppressor pathway is an early obligatory event in human cancer. One of the key targets of the Rb pathway is the family of E2F transcription factors, which regulate cell proliferation and apoptosis. Overlapping and redundant functions of mammalian members of the E2F and Rb tumor suppressor protein (pRB) families is a major hurdle in identification of their in vivo function. Model organisms such as Drosophila provide attractive alternatives to complement studies in mammalian systems due to high conservation of the Rb pathway and the amenability of flies to genetic analysis. Our research program is aimed at addressing two fundamental questions: what are the essential functions of the Rb pathway in development, and how do Rb pathway mutations affect different cell types in the tissue? To address these questions, we are using genetic, genomic and single-cell RNA-seq approaches. We have identified adult skeletal muscle where E2F and Rb proteins regulate growth and myofibrillogenesis, which is important for viability. We will determine how the Rb pathway regulates gene expression and cellular metabolism during muscle development, and will investigate the systemic effects inferred by E2F deficiency in specific tissues such as adult skeletal muscle and the fat body. We have adapted Drop-seq, a single-cell RNA-sequencing technology, to determine the effect of Rb pathway mutations in heterogeneous tissue. We have established the feasibility of this approach by building the first cell atlas of the Drosophila wild- type eye imaginal disc. Profiling of Rb mutants by Drop-Seq revealed elevated intracellular acidification in a small cell population that makes them uniquely sensitive to apoptosis. The link between apoptosis and acidification will be evaluated further. Among other questions to be addressed with Drop-seq is the investigation of why inactivation of the Hippo pathway induces dedifferentiation of Rb mutant photoreceptors. Collectively, the proposed research will enhance our knowledge of pRB and its functions during animal development.